Anthony H. OLAVESEN and Peter GACESA*. Department of ..... Tavazzi L, Heyndriekx GR, Ray M,. Chiariello M, Distante A, Askenazi J, Salerno J, Carpentier J,.
Bioscience Reports 5, 329-334 (1985) Printed in Great Britain
329
Preferential uptake of intravenously administered hyaluronidase (Hyalosidase) by damaged rat myocardium
3ulia S. EARNSHAW, Kheng H. PEH l, Kenneth S. DODGSON, Anthony H. OLAVESEN and Peter GACESA* Department of Biochemistry, University College, P.O. Box 78, Cardiff CFI IXL, Wales, U.K.; and IBiorex Laboratories Ltd., Biorex House, Canonbury Villas, London N1 2HB, U.K. (Received 9 April 1985)
The i n d u c t i o n of m y o c a r d i a l i n f a r c t i o n in rats by l i g a t i o n of the l e f t - a n t e r i o r c o r o n a r y a r t e r y was confirmed by measurement of increased plasma levels of c r e a t i n e k i n a s e , a s p a r t a t e aminotransferase and l a c t a t e dehydrogenase. Using this model system it has been e s t a b l i s h e d t h a t i n t r a v e n o u s administration of 1 2 S I - l a b e l l e d h y a l u r o n i d a s e to r a t s r e s u l t e d in a preferential u p t a k e of t h e e n z y m e by d a m a g e d myocardium as compared to normal heart tissue. M a m m a l i a n h y a l u r o n i d a s e s ( h y a l u r o n a t e #-glycanohydrolase, EC 3.2.1.35) are endoglycosidases that can hydrolyse the fl(1§ glycosidic bonds of D-giucuronic acid-containing glycosaminoglycans. The most studied type of hyaluronidase has been obtained from bovine testes (Borders & Raftery, 1968; Pope et al., 1976; Lyon & Phelps, 1981) although a different form of the enzyme is located in the lysosomes of other mammalian tissues (Meyer, 1971). R e l a t i v e l y c r u d e preparations of bovine testicular hyaluronidase have been used t h e r a p e u t i c a l l y in t h e t r e a t m e n t of myocardial i n f a r c t i o n ( M a r o k o et al., 1977). More recently, a U.K. based, m u l t i - c e n t r e c l i n i c a l t r i a l has been carried out to evaluate the e f f i c a c y of a highly purified preparation of hyaluronidase in the t r e a t m e n t of myocardial infarction (Flint et al., 1982; Henderson et al., 1982; Saltissi et al., 1982). This particular preparation, which is known c o m m e r c i a l l y as GL e n z y m e or Hyalosidase, significantly r e d u c e s t h e c u m u l a t i v e m o r t a l i t y of p a t i e n t s m e a s u r e d over a six-month follow-up period. Although it has been shown that hyaluronidase has a beneficial e f f e c t on p a t i e n t s with myocardial infarction there is very little conclusive evidence to explain how the enzyme functions therapeutically. O t h e r groups (Wolf et al., 1981; Earnshaw et al., 1985), including our own, have demonstrated that the bulk of the hyaluronidase administered intravenously to animals is rapidly accumulated by To whom reprint requests should be sent.
330
EARNSHAW ET AL.
t h e liver. H y a l u r o n i d a s e is a high-mannose type of glycoprotein (Michalski et a l , 1984) and the accumulation of the enzyme by the liver can be decreased by the pre-administration of mannose-containing compounds (Earnshaw et a l , 1985). Only a small proportion of the total administered hyaluronidase reaches the rat myocardium but this may reflect the fact that the heart tissue is undamaged in these animals. The objective of the present work was to establish whether d a m a g e d r a t m y o c a r d i u m has an ehnanced ability to accumulate hyaluronidase. Materials
and Methods
Chemicals
A highly purified preparation of bovine testicular hyaluronidase (>40000 i.u./mg) was kindly donated by Biorex Laboratories~ London~ U . K , and was the same product (GL enzyme) that has been used in clinical trials in the U.K. Potassium hyaluronate for the routine assay of activity was supplied by Miles Laboratories, Stoke Poges~ U.K. All other biochemicals and chemicals were purchased either from BDH Chemicals, Poole, U.K. or from the Sigma Chemical Co., Poole~ U.K. Measurement
of enzyme activities
Hyaluronidase activity was estimated by the measurement of the N-acetyl D-glucosamine reducing end groups released by the action of the enzyme on potassium hyaluronate (Gacesa et a l , 1981). The assay procedure was calibrated by reference to the W.H.O. International Standard for hyaluronidase (Humphrey~ 1957). A s p a r t a t e aminotransferase (Bergmeyer & Bernt~ 1974)~ lactate dehydrogenase (Morgenstern et al., 1965) and creatine kinase (Rosalki~ 1967) were a s s a y e d using a u t o m a t e d methods. To quantify the creatine kinase in the myocardium~ samples of tissue were sonicated prior to assay (Kjekshus & Sobe% 1970). Occlusion
of the rat left anterior coronary
artery
In order to p r o d u c e a l i m i t e d myocardial infarction the left anterior coronary artery was ligated (Camilleri et a l , 19gl). Female rats of approx. 200 g body weight were anaesthetized and the thorax of each was shaved. The thorax was opened by transecting the left sixth rib, the heart was lifted without damage~ and the left anterior coronary artery was ligated. The heart was replaced in the thorax and any air was forced out of the cavity prior to closure. Sham operations were also carried out on a similar numbers of rats which then served as controls for each experiment. The sham operation was identical to that used when actual occlusion was carried out, except that the ligature was simply passed under the appropriate artery but not tied. Animals were left for either 4 h or 24 h before administration of hyaluronidase. In vivo experiments
The methods for the preparation of enzymically active 12SI-labelIed hyaluronidase~ the intravenous administration of the enzyme and the estimation of the radioactivity in the various rat organs and tissues have been described previously (Earnshaw et al.~ 1985). 12SI-Labelled
HYALURONIDASE
UPTAKE
BY DAMAGED
MYOCARDIUM
331
hyaluronidase (175 i.u., 1.7 ~Ci) was a d m i n i s t e r e d to the rat via the jugular vein, and a f t e r 10 min a blood s a m p l e was t a k e n from the dorsal a o r t a t h a t resulted in the d e a t h of the animal. The serum d e r i v e d f r o m the sample was subsequently used to e s t i m a t e the levels o f c i r c u l a t i n g c r e a t i n e kinase, l a c t a t e d e h y d r o g e n a s e and a s p a r t a t e aminotransferase activities. Remaining blood was r e m o v e d from the rat by infusion ( p e r i s t a l t i c pump) of a g l u c o s e / c i t r a t e solution (see Earnshaw et al., 1985) and the organs were then d i s s e c t e d out prior to e s t i m a t i o n of r a d i o a c t i v e c o n t e n t by 3'-counting. The two sides of the h e a r t were t r e a t e d s e p a r a t e l y . R e s u l t s and D i s c u s s i o n Ligation of the l e f t a n t e r i o r c o r o n a r y a r t e r y of rats r e s u l t e d in a d e p l e t i o n of the blood supply to the l e f t - h a n d side of the h e a r t . In t h e s e animals the l e f t - h a n d side ( o c c l u d e d ) of the h e a r t was paler in colour than the right and could be distinguished c l e a r l y post m o r t e m . One of the parameters u s e d in clinical medicine to diagnose m y o c a r d i a l i n f a r c t i o n is the m e a s u r a b l e r e l e a s e of c e r t a i n h e a r t - m u s c l e cytoplasmic e n z y m e s into the serum (Strandjord & Clyson, 197#). C o n s e q u e n t l y , levels of a s p a r a t e a m i n o t r a n s f e r a s e , l a c t a t e d e h y d r o genase and c r e a t i n e kinase were m e a s u r e d in serum samples from rats to ascertain w h e t h e r d a m a g e t o t h e m y o c a r d i u m had o c c u r r e d . Results ( T a b l e 1) showed t h a t all t h r e e e n z y m e s w e r e e l e v a t e d in the animals with an occluded a r t e r y as c o m p a r e d to the s h a m - o p e r a t e d group. A s p a r t a t e a m i n o t r a n s f e r a s e a c t i v i t y was significantly higher in the t e s t animals at both # h and 2# h a f t e r occlusion, whereas l a c t a t e d e h y d r o g e n a s e a c t i v i t y was higher at both t i m e s but only significantly so a t # h. Although c r e a t i n e kinase a c t i v i t y was e l e v a t e d in the t e s t animals the resuits did not r e a c h s t a t i s t i c a l s i g n i f i c a n c e a t e i t h e r g h or 2# h a f t e r occlusion of the a r t e r y .
Table I.
Levels of enzymes appearing in the serum of rats with occluded myocardia
Results were expressed as m e a ns • S.D. (no. of e x p e r i m e n t s ) . Probability values have been calculated using Student's paired t-test. Analysis of the results by the non-parametric method of Mann and Whitney gave similar probability values. Enzyme activity (i.u./l) Aspartate aminotransferase Normals
4 h Occluded 4 h Sham-operated
24 h Occluded 24 h Sham-operated
86 • 6
(4)
Lactate dehydrogenase 639 • 389
(4)
Creatine kinase 85 • 40
813 • 246 (4) P
